Patterned flow-cells useful for nucleic acid analysis

ABSTRACT

Provided is a surface having metal regions and an interstitial region having a composition that differs from the metal regions, wherein a continuous gel layer coats the surface across the metal regions and the interstitial regions. Nucleic acids or other analytes can be attached to the continuous gel layer such that a greater amount is attached over the metal regions than over the interstitial region. Also provided are methods for making such surfaces. Methods are also provided for making an array of nucleic acids or other analytes using such surfaces.

This patent application is a continuation of U.S. patent applicationSer. No. 13/492,661 filed on Jun. 8, 2012 which claims priority to U.S.Provisional Patent Application No. 61/495,266 filed on Jun. 9, 2011,each of which is incorporated herein by reference in its entirety.

BACKGROUND

The present invention relates generally to genomics analysis, and morespecifically to methods for producing arrays for high throughputgenomics analysis.

The task of cataloguing human genetic variation and correlating thisvariation with susceptibility to disease stands to benefit from advancesin genome wide sequencing methodologies. This cataloguing effort holdsthe promise for identifying the markers in each person's genome thatwill help medical professionals determine susceptibility of theindividual to disease, responsiveness to specific therapies such asprescription drugs, susceptibility to dangerous drug side effects andother medically actionable characteristics. The cataloguing effort iswell under way. This is due in large part to commercially availablegenome sequencing methodologies which are sufficiently cost effective toallow test subjects to be evaluated in a research setting. Improvementsin sequencing methodologies are needed to accelerate the cataloguingeffort. Perhaps even more significant is that the relatively high costof sequencing has hindered the technology from moving beyond theresearch centers and into the clinic where doctors can obtain sequencesfor the general population.

Sequencing methodologies and the systems used to carry them out, exploita complex collection of technologies. Improvements in some of thesetechnologies have been shown to provide substantial cost reductions.However, it is difficult to predict which if any is amenable to costreducing improvements. Given the dependencies between the technologiesin the sequencing systems it is even more difficult to predict which canbe modified without having an adverse impact on the overall performanceof the methodology. Thus, there exists a need to identify improvementsin sequencing methodologies and systems that can bring the promise ofgenomics research to the clinic where lives can be improved and in manycases saved. The present invention satisfies this need and providesrelated advantages as well.

BRIEF SUMMARY

The present disclosure provides a method of preparing a surface. Themethod can include the steps of (a) providing a surface having metalregions and interstitial regions, the interstitial regions having acomposition that differs from the metal regions; (b) contacting thesurface with a fluid including a polymerizable material, wherein thefluid coats the surface across the metal regions and the interstitialregions; and (c) polymerizing the polymerizable material, therebyforming a continuous gel layer that coats the surface across the metalregions and the interstitial regions, wherein portions of the gel layerthat coat the metal regions have greater mass than portions of the gellayer that coat the interstitial regions.

In particular embodiments, a method of making a nucleic acid array isprovided. The method can include the steps of (a) providing a surfacehaving metal regions and interstitial regions, the interstitial regionshaving a composition that differs from the metal regions, wherein acontinuous gel layer coats the surface across the metal regions and theinterstitial regions; and (b) contacting the continuous gel layer with afluid including nucleic acids under conditions wherein the nucleic acidsbecome attached to the gel layer and wherein a greater amount of thenucleic acids from the fluid attach to portions of the gel layer thatcoat the metal regions than the amount of nucleic acids that attach toportions of the gel layer that coat the interstitial regions.

This disclosure provides a method of making a nucleic acid array. Inparticular embodiments, the method includes the steps of (a) providing asurface having metal regions and interstitial regions, the interstitialregions having a composition that differs from the metal regions,wherein a continuous gel layer coats the surface across the metalregions and the interstitial regions; (b) contacting the continuous gellayer with a fluid comprising nucleic acids; and (c) selectivelymodifying the portions of the gel layer that coat the interstitialregions compared to the portions of the gel layer that coat the metalregions, thereby attaching the nucleic acids to the gel layer, wherein agreater amount of the nucleic acids from the fluid attach to portions ofthe gel layer that coat the metal regions than the amount of nucleicacids that attach to portions of the gel layer that coat theinterstitial regions.

Methods are provided for preparing a surface, including the steps of (a)providing a surface having mask regions and transparent regions, themask regions having a composition that blocks transmittance of radiationin a first part of the electromagnetic radiation spectrum andtransparent regions having a composition that transmits radiation in thefirst part of the electromagnetic radiation spectrum; (b) contacting thesurface with a fluid comprising a photo-polymerizable material, whereinthe fluid coats the surface across the mask regions and the transparentregions; and (c) selectively photo-polymerizing the portions of thefluid that coat the transparent regions compared to the portions of thefluid that coat the mask regions, thereby forming a gel that coats thetransparent regions.

Also provided is a method of making a nucleic acid array. The method caninclude the steps of (a) providing a surface having mask regions andtransparent regions, the mask regions having a composition that blockstransmittance of radiation in a first part of the electromagneticspectrum and transparent regions having a composition that transmitsradiation in the first part of the electromagnetic radiation spectrum,wherein a continuous gel layer coats the surface across the mask regionsand the transparent regions; (b) contacting the continuous gel layerwith a fluid including nucleic acids under conditions wherein thenucleic acids become attached to the gel layer and wherein a firstsubpopulation of the nucleic acids attach to portions of the gel layerthat coat the mask regions and a second subpopulation of nucleic acidsattach to portions of the gel layer that coat the transparent regions;and (c) irradiating the surface with radiation in the first part of theelectromagnetic spectrum, thereby selectively modifying the nucleicacids of one of the subpopulations compared to the nucleic acids of theother subpopulation.

This disclosure further provides a method for preparing a surface thatincludes the steps of (a) providing a surface having mask regions andtransparent regions, the mask regions having a composition that blockstransmittance of radiation in a first part of the electromagneticradiation spectrum and transparent regions having a composition thattransmits radiation in the first part of the electromagnetic radiationspectrum, the surface further including photo-reactive crosslinkingreagents attached thereto; (b) contacting the surface with a fluidcomprising a photo-polymerizable material, wherein the fluid coats thesurface across the mask regions and the transparent regions; and (c)selectively irradiating the portions of the fluid that coat thetransparent regions compared to the portions of the fluid that coat themask regions, wherein the portions of the fluid that coat thetransparent regions are photo-polymerized to form a gel and wherein thegel is photo-crosslinked to the surface at the transparent regions.

This disclosure further provides a nucleic acid array. The array caninclude a surface having metal regions and interstitial regions having acomposition that differs from the metal regions, wherein a continuousgel layer coats the surface across the metal regions and theinterstitial regions, wherein nucleic acids are attached to thecontinuous gel layer, and wherein a greater amount of the nucleic acidsare attached to portions of the gel layer that coat the metal regionsthan the amount of nucleic acids that attach to portions of the gellayer that coat the interstitial region.

In particular embodiments, a nucleic acid array can have a surface with(a) mask regions or metal regions, wherein individual mask regions areattached to a single nucleic acid template; and (b) interstitialregions, wherein a continuous gel layer coats the surface across themask regions and the interstitial regions, wherein a plurality ofnucleic acid copies of the template nucleic acid are attached to thecontinuous gel layer in respective clusters surrounding the metalregions. The mask regions or metal regions can have a composition thatblocks transmittance of electromagnetic radiation in a first part of theelectromagnetic radiation spectrum and the interstitial regions can havea composition that transmits radiation in the first part of theelectromagnetic radiation spectrum.

Also provided is a nucleic acid array having a surface with mask regionshaving a composition that blocks transmittance of electromagneticradiation in a first region of the electromagnetic radiation spectrumand transparent regions having a composition that transmits radiation inthe first part of the electromagnetic radiation spectrum, wherein acontinuous gel layer coats the surface across the mask regions and thetransparent regions, wherein nucleic acids are attached to thecontinuous gel layer, and wherein a greater amount of the nucleic acidsare attached to portions of the gel layer that coat the transparentregions than the amount of nucleic acids that are attached to portionsof the gel layer that coat the mask regions.

Further provided is a nucleic acid array having a surface with maskregions having a composition that blocks transmittance ofelectromagnetic radiation in a first part of the electromagneticradiation spectrum and transparent regions having a composition thattransmits radiation in the first part of the electromagnetic radiationspectrum, wherein a continuous gel layer coats the surface across themask regions and the transparent regions, wherein nucleic acids areattached to the continuous gel layer, and wherein a greater amount ofthe nucleic acids are attached to portions of the gel layer that coatthe mask regions than the amount of nucleic acids that are attached toportions of the gel layer that coat the transparent regions.

The methods and compositions are exemplified above in the context ofembodiments that use a nucleic acid as an analyte. This is done forpurposes of illustration and is not intended to be limiting. Rather anyof a variety of analytes can be used in place of nucleic acids in theexamples set forth above and throughout this disclosure. Exemplaryanalytes are set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows diagrammatic representations of methods for making metalpatterned surfaces using a combination of photolithography and metallayer deposition including a lift-off method (A), and an etch method(B).

FIG. 2 shows diagrammatic representations of (A) a top view of a surfacehaving metal regions and (B) a side view of the surface after coatingwith a continuous gel layer, grafting of primers and growth of DNAclusters on the primers.

FIG. 3 shows (A) photographs of a full view for a flow-cell having ametal patterned surface and a magnified view of a portion of thesurface; (B) a fluorescent image of a metal patterned surface aftercoating with silane-free acrylamide (SFA) polymer and grafting ofprimers to the SFA polymer and hybridization of fluorescent labeledprobes to the primers, and a plot of grayscale vs. distance for a linemeasured across the surface; (C) and (D) high magnification images ofDNA clusters grown on a metal patterned surface and a plot of grayscalevs. distance for a line measured across the surface.

FIG. 4 shows results of selectively photo-cleaving primers at non-metalregions of a metal patterned flow-cell: (A) experimental set up forirradiating the flow-cell with UV light from the bottom of theflow-cell; (B) image of the flow-cell after UV exposure; (C) plot ofgrayscale vs. distance showing primer distribution for lane 8 of theflow-cell.

FIG. 5 shows a schematic representation of two methods for creating geland/or nucleic acid features on a metal patterned flow-cell.

FIG. 6 shows a schematic representation of a method for photo-initiationof SFA polymerization in interstitial regions of a metal patternedsurface and selective DNA cluster growth in the interstitial regionscompared to metal regions.

FIG. 7 shows a schematic representation of a method for photo-cleavingof primers in the interstitial regions of a metal patterned surface andselective DNA cluster growth in the metal regions compared to theinterstitial regions.

FIG. 8 shows a synthetic scheme for attaching a photo-reactivecrosslinking reagent to a surface and photo-crosslinking a gel to thesurface.

FIG. 9 shows a surface having a photo-reactive crosslinking reagent attransparent regions patterned on a surface and a photoreaction tocrosslink a gel to the surface at the transparent regions.

FIG. 10 shows a synthetic scheme for a photo-polymerizable material.

DETAILED DESCRIPTION

The disclosure provides methods for coating a surface with a gel.Surprisingly, using methods set forth herein a greater mass of gel canbe formed over a metal surface (such as a metal oxide) relative to themass formed over another surface. Thus a method is provided in which asurface having a pattern of metal regions and interstitial regions iscontacted with a polymerizable material and the material is polymerizedto form a continuous gel layer that coats the surface such that portionsof the gel layer that coat the metal regions have greater mass thanportions of the gel layer that coat the interstitial regions.

This disclosure also provides methods for making an array of moleculesattached to a surface. Particular embodiments exploit the surprisingobservation that a larger quantity of molecules can be attached to aportion of a gel layer that coats a metal surface relative to thequantity of molecules attached to a similar gel layer coating othersurfaces. According to methods set forth in further detail below, asurface having a pattern of metal regions and interstitial regionscovered by a continuous gel layer can be treated to attach molecules tothe gel layer, the result of which is an array of the molecules presentas features, the features corresponding to the portions of the gel layerthat cover the metal regions.

In particular embodiments, a surface made in accordance with the methodsset forth herein can be irradiated such that the metal regions act as amask protecting portions of a gel layer, or molecules at the portions ofthe gel layer, that coat the metal regions. In contrast, portions of thegel layer that cover the interstitial regions, or molecules at thoseportions, are modified by the irradiation. For example, the gel layercan be irradiated to produce an array of gel portions having desiredproperties and corresponding to the shape and location of the metalregions. Similarly, the irradiation can be used to selectively ablate orremove molecules in the interstitial regions to leave an array ofmolecules corresponding to the shape and location of the metal regions.Metal oxides such as indium tin oxide and zinc oxide are particularlyuseful materials for a mask because the band gap for these masks is suchthat they absorb radiation in the UV range and transmit radiation in thevisible range. Thus, metal oxide can form a mask that is useful in aprocess of making or modifying an array using UV range photo-reactions,but will not interfere with analytical applications of the array usingdetection in the visible range.

Embodiments of the invention are exemplified and described herein withreference to metal regions and interstitial regions that are located ona surface. However, the invention need not be limited such that theregions are on a surface. Rather in particular embodiments, a metalregion, interstitial region or both can occur in a solid support orunder the surface of the solid support. Furthermore, the location of ametal region, interstitial region or both can change with respect to asolid support. The change can be brought about by modifying the solidsupport, for example, by etching or polishing the solid support to bringa region to the surface from below. The change can be brought about bycovering a region on a surface, by coating the surface or by building upthe solid support.

Although several aspects of the invention are exemplified with respectto the use of metal as a mask, it will be understood that othermaterials appropriate to block radiation of a particular wavelength canalso be used. Generally, a mask region on a surface, such as a metalregion on a surface, can provide a near-field mask to selectively blockirradiation of a particular wavelength. The near-field mask is generallylocated in a plane that is less than 1 wavelength from the surface (inthe z-dimension) and therefore provides advantages in overcomingdiffraction limits of other masking methods, such as those used instandard lithography techniques. Surprisingly, it has been observed thatin the methods set forth herein a near-field mask is capable ofprotecting a gel layer and molecules attached to the gel layer from theeffects, not only of collimated radiation, but also of non-collimatedradiation. As set forth in further detail below, a near-field mask thatis on a surface and used during an irradiation step for preparing thesurface, or materials in contact with the surface, can be subsequentlyremoved from the surface. Alternatively, the near-field mask can beretained on the surface for one or more other manipulation including,but not limited to, those manipulations set forth herein. The mask mayinfluence one or more other manipulations carried out on or near thesurface, but need not have any particular impact for any particularmanipulation.

As used herein, the term “surface” is intended to mean an external partor external layer of a solid support. The solid support can be a rigidsolid and optionally can be impermeable to liquids or gases. The solidsupport can also be a semi-rigid solid, for example, being permeable toliquids or gases. The surface can be in contact with another materialsuch as a gas, liquid, gel, second surface of a similar or differentsolid support, metal, or coat. The surface, or regions thereof, can besubstantially flat. The surface can have surface features such as wells,pits, channels, ridges, raised regions, pegs, posts or the like.

As used herein, the term “metal region” is intended to mean an area in asubstrate or on a surface that contains a metal. A metal region candiffer from another region with respect to the type of metal or quantityof a particular metal that is present at the metal region. The metalregion can have a continuous coating of one or more type of metal. Themetal region can have a composition and thickness that is sufficient tomask the transmittance of electromagnetic radiation from a particularpart of the electromagnetic radiation spectrum including, for example, apart that is in the radio, microwave, infrared, visible, ultraviolet,X-ray or gamma ray parts of the spectrum. The metal can have a thicknessin the range of 1 atom thickness to the thickness of a flow cell orother chamber where the metal coated surface resides. For example, thethickness of the metal can be at least about 1 nm, 5 nm, 10 nm, 50 nm,100 nm, 250 nm or 500 nm. Alternatively or additionally, the thicknessof the metal can be at most about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 250nm or 500 nm. The metal region can include, for example, aluminum,aluminum oxide, titanium, titanium oxide, zinc, zinc oxide, zincsulfide, zinc selenide, boron or indium tin oxide. The metal region caninclude a Group I (alkali) metal, examples of which include, lithium(Li), potassium (K), rubidium (Rb), caesium (Cs) or francium (Fr). Themetal region can include a Group II (alkaline earth metal) such asberyllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium(Ba) or radium (Ra). Also useful as a metal region are those thatinclude a heavy metal halide, polycrystalline material such as lanthanumoxides, arsenic trisulfide, amorphous/polycrystalline silicon,silicon/germanium alloy, III-V semiconductor such as GaAs, or II-VIsemiconductor such as CdS. Metals that form a dielectric stack or smallinterference mirror can be useful. In particular embodiments, a metalthat is present at a metal region is positively charged. As evident fromthe examples above, a metal region can include a metal oxide.

As used herein, the term “mask region” is intended to mean an area in asubstrate or on a surface having a composition and thickness that issufficient to block the transmittance of electromagnetic radiation froma particular part of the electromagnetic radiation spectrum. In some butnot all embodiments, the substrate or surface also has an interstitialregion that is transparent to at least a portion of the particular partof the electromagnetic radiation spectrum that is blocked by the mask.The part of the electromagnetic radiation spectrum can include, forexample, a part that is in the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. An exemplary maskregion is a “near-field mask region” which is located such that theplane of the mask that is distal to the surface is less than about 1wavelength distance from the surface (in the z-dimension). In particularembodiments a near field mask is used with electromagnetic radiation inthe parts of the spectrum set forth above. Accordingly, the plane of themask that is distal to the surface can be less than about 10 nm, 50 nm,100 nm, 200 nm, 300 nm, 400 nm, or 500 nm from the surface. Inparticular embodiments, a metal region can function as a mask region oras a near-field mask region. As such, a mask region or near-field maskregion can have a composition exemplified herein for metal regions.

As used herein, the term “interstitial region” is intended to mean anarea in a substrate or on a surface that separates other areas of thesubstrate or surface. The term can refer to an area that separates otherareas of a surface that are occupied by one or more feature. Forexample, an interstitial region can separate one feature from anotherfeature. Thus, a first metal region on a surface can be separated from asecond metal region on the surface by an interstitial region. The tworegions that are separated from each other can be discrete, lackingcontact with each other. In another example, an interstitial region canseparate a first portion of a feature from a second portion of afeature. Accordingly, two finger-like projections of a metal region canbe separated by an interstitial region or an interstitial region can bethe hole that separates portions of a donut-shaped metal region. Asillustrated by the above examples, the separation provided by aninterstitial region can be partial or full separation. An interstitialregion can have a composition that is transparent to electromagneticradiation from a particular part of the electromagnetic radiationspectrum including, for example, a part that is in the radio, microwave,infrared, visible, ultraviolet, X-ray or gamma ray parts of thespectrum.

As used herein, the term “gel” is intended to mean a semi-rigid solidthat is permeable to liquids and gases. Exemplary gels include, but arenot limited to those having a colloidal structure, such as agarose;polymer mesh structure, such as gelatin; or cross-linked polymerstructure, such as polyacrylamide.

As used herein, the term “continuous,” when used in reference to a layerthat coats a surface across two or more regions, is intended to meanthat the layer bridges the two or more regions. It will be understoodthat the layer may be composed of a material that is porous or that hasstructural interruptions, so long as the material forms a layer thatbridges the two or more regions.

As used herein, the term “coat,” when used as a verb, is intended tomean providing a layer or covering on a surface. As a result at least aportion of the surface can have a layer or cover. In some cases theentire surface can have a layer or cover. In alternative cases only aportion of the surface will have a layer or covering. The term “coat,”when used to describe the relationship between a surface and a material,is intended to mean that the material is present as a layer or cover onthe surface. The material can seal the surface, for example, preventingcontact of liquid or gas with the surface. However, the material neednot form a seal. For example, the material can be porous to liquid, gas,or one or more components carried in a liquid or gas. Exemplarymaterials that can coat a surface include, but are not limited to, a gelsuch as polyacrylamide or agarose, liquid, metal, a second surface,plastic, silica, or gas.

As used herein, reference to “selectively” manipulating a first thingcompared to second thing is intended to mean that the manipulation has agreater effect on the first thing compared to the effect on the secondthing. The manipulation need not have an effect on the second thing. Themanipulation can have an effect on the first thing that is at least 1%,5%, 10%, 25%, 50%, 75%, 90%, 95%, or 99% greater than the effect on thesecond thing. The manipulation can have an effect on the first thingthat is at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 100 fold,1×10³ fold, 1×10⁴ fold or 1×10⁶ fold higher than the effect on thesecond thing. The manipulation can include, for example, modifying,contacting, treating, changing, cleaving (e.g. of a chemical bond),photo-chemically cleaving (e.g. of a chemical bond), forming (e.g. of achemical bond), photo-chemically forming (e.g. of a chemical bond),covalently modifying, non-covalently modifying, destroying,photo-ablating, removing, synthesizing, polymerizing,photo-polymerizing, amplifying (e.g. of a nucleic acid), copying (e.g.of a nucleic acid), extending (e.g. of a nucleic acid), ligating (e.g.of a nucleic acid), or other manipulation set forth herein or otherwiseknown in the art.

As used herein, the prefixes “photo” or “photo-” mean relating to lightor electromagnetic radiation. The term can encompass all or part of theelectromagnetic spectrum including, but not limited to, one or more ofthe ranges commonly known as the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. The part of thespectrum can be one that is blocked by a metal region (or other maskregion) of a surface such as those metals set forth herein.Alternatively or additionally, the part of the spectrum can be one thatpasses through an interstitial region of a surface such as a region madeof glass, plastic, silica, or other material set forth herein. Inparticular embodiments, radiation can be used that is capable of passingthrough a metal. Alternatively or additionally, radiation can be usedthat is masked by glass, plastic, silica, or other material set forthherein.

The present disclosure provides a method of preparing a surface. Themethod can include the steps of (a) providing a surface having metalregions and interstitial regions, the interstitial regions having acomposition that differs from the metal regions; (b) contacting thesurface with a fluid including a polymerizable material, wherein thefluid coats the surface across the metal regions and the interstitialregions; and (c) polymerizing the polymerizable material, therebyforming a continuous gel layer that coats the surface across the metalregions and the interstitial regions, wherein portions of the gel layerthat coat the metal regions have greater mass than portions of the gellayer that coat the interstitial regions.

A surface that is used in accordance with the methods set forth hereincan be present on any of a variety of substrates. The surface can belocated on a substrate or material that provides a solid or semi-solidfoundation for a mask region or a metal region. Exemplary types ofsubstrate materials include glass, modified glass, functionalized glass,inorganic glasses, microspheres, including inert and/or magneticparticles, plastics, polysaccharides, nylon, nitrocellulose, ceramics,resins, silica, silica-based materials, carbon, metals, an optical fiberor optical fiber bundles, polymers and multiwell (e.g. microtiter)plates. Specific types of exemplary plastics include acrylics,polystyrene, copolymers of styrene and other materials, polypropylene,polyethylene, polybutylene, polyurethanes and Teflon™. Specific types ofexemplary silica-based materials include silicon and various forms ofmodified silicon.

In particular embodiments the substrate provides a support for creationof features having attached biopolymers, including nucleic acids,polypeptide and/or other polymers. Accordingly, substrates employed inthe art as microarrays are particularly useful. Exemplary substrates arethose used for a Sentrix® Array or Sentrix® BeadChip Array availablefrom Illumina®, Inc. (San Diego, Calif.) or those described in U.S. Pat.Nos. 6,266,459; 6,355,431; 6,770,441 and 6,859,570 and PCT PublicationNo. WO 00/63437 (each of which is incorporated by reference in itsentirety). Other arrays having useful substrates include those set forthin US Pat. Pub. Nos. 2005/0227252 A1, 2006/0023310 A1, 2006/006327 A1,2006/0071075 A1, 2006/0119913 A1, U.S. Pat. Nos. 6,489,606; 7,106,513;7,126,755; 7,164,533; and PCT Pub. Nos. WO 05/033681 and WO 04/024328(each of which is hereby incorporated by reference in its entirety).

Further examples of commercially available microarrays having substratesthat can be used include, for example, an Affymetrix GeneChip®microarray or other microarray such as those described, for example, inU.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074;5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219;5,968,740; 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860;6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831;6,416,949; 6,428,752 and 6,482,591 (each of which is hereby incorporatedby reference in its entirety).

Substrates used in the manufacture of spotted microarrays can also beused. An exemplary spotted microarray is a CodeLink™ Array availablefrom Amersham Biosciences. Another microarray made from substrates thatcan be useful in the invention is one that is manufactured using inkjetprinting methods such as SurePrint™ Technology available from AgilentTechnologies. Other substrates include, but are not limited to, thosedescribed in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211;5,658,734; 5,837,858; 5,919,523; 6,287,768; 6,287,776; 6,288,220;6,297,006; 6,291,193; and 6,514,751 and PCT Pub. Nos. WO 93/17126 and WO95/35505 (each of which is hereby incorporated by reference in itsentirety).

Those skilled in the art will know or understand that the compositionand geometry of a substrate can vary depending on the intended use andpreferences of the user. Therefore, although planar substrates such asslides, chips or wafers are exemplified herein for illustration, giventhe teachings and guidance provided herein, those skilled in the artwill understand that a wide variety of other substrates exemplifiedherein or well known in the art also can be used in the methods and/orcompositions of the invention.

Generally, a surface that is used in a method or composition set forthherein will be planar. However, the surface need not be planar and caninstead have surface features such as wells, pits, channels, ridges,raised regions, pegs, posts or the like. The surface can be porous ornonporous to suit particular applications. A metal region or patch canbe located in or on a particular surface feature such as those set forthabove. Similarly an interstitial region can be located in or on aparticular surface feature. Furthermore, these or other surface featurescan occur within a metal region, mask region or interstitial region.

In particular embodiments, a surface or region thereof can be located ina vessel such as a well, tube, channel, cuvette, Petri plate, bottle orthe like. A particularly useful vessel is a flow-cell, for example, asdescribed in US Pat. Pub. No. 2010/0111768 A1 or Bentley et al., Nature456:53-59 (2008), each of which is incorporated herein by reference inits entirety. Exemplary flow-cells are those that are commerciallyavailable from Illumina, Inc. (San Diego, Calif.). Another particularlyuseful vessel is a well in a multiwell plate or microtiter plate.

A surface can have one or more regions. The regions can be distinguishedby characteristics such as composition of the region, shape of theregion, size of the region, thickness of the region (e.g. in thez-dimension), location of the region on the surface (e.g. in the x or ydimension). Accordingly, two or more different regions can bedistinguished based on differences in one or more characteristics.

Exemplary compositions for one or more regions include metals, such asthose set forth elsewhere herein; substrate materials, such as those setforth elsewhere herein; mask materials that prevent passage of radiationin a particular range of the electromagnetic spectrum, examples of whichare set forth elsewhere herein; optical filter materials that pass lightin a certain wavelength range, outside of a certain wavelength range,above a certain cutoff wavelength or below a certain cutoff wavelength;non-metallic materials; or chemically reactive compositions such asthose capable of attaching to a molecule of interest. In particularembodiments the reactive compositions are molecular linkers capable ofcovalently bonding to a molecule of interest (e.g. photo-reactivecrosslinking reagents), binding agents capable of binding to anothermolecule via non-specific interactions (e.g. hydrogen bonding, ionicbonding, van der Waals interactions etc.) or via specific interactions(e.g. affinity interactions, receptor-ligand interactions,antibody-epitope interactions, avidin-biotin interactions,streptavidin-biotin interactions, lectin-carbohydrate interactions,etc.).

A region on a surface can have any of a variety of geometric shapes.Examples include, without limitation, rectangular, square, circular,elliptical, oval, triangular, polygonal, trapezoidal or irregularshapes. Several regions can be present on a surface in the form of anarray. For example, the array of regions can appear as a grid of spotsor patches. Regions that form an array can be located in a repeatingpattern or in an irregular non-repeating pattern. Particularly usefulpatterns are hexagonal patterns, rectilinear patterns, grid patterns,patterns having reflective symmetry, patterns having rotationalsymmetry, or the like. Asymmetric patterns can also be useful. The pitchcan be the same between different pairs of nearest neighbors or thepitch can vary between different pairs of nearest neighbors. Generally,two or more regions on a surface are separated by interstitial regions.The separation can be partial such that an interstitial region separatespart of one region from part of another region. Alternatively, theseparation can be sufficiently complete to render two regions discretefrom each other. Thus, at least one metal region or mask region on asurface can be completely surrounded by at least a portion of aninterstitial region.

The size of a region on a surface can be selected to suit a particularapplication. For example, in some embodiments a region can have a sizethat accommodates only a single nucleic acid molecule. A surface havinga plurality of features in this size range is useful for constructing anarray of molecules for detection at single molecule resolution. Features(e.g. metal features or mask features) in this size range are alsouseful for capture of a single nucleic acid template molecule to seedsubsequent formation of a homogenous colony, for example, via bridgeamplification. In this example, the bridge amplification can be primedby primer nucleic acids that are attached to a gel layer that is incontact with a metal feature or mask feature, the feature being attachedto a single nucleic acid template. Thus, the feature can seed growth ofa cluster of nucleic acid copies of the template that forms in the gellayer around the feature.

Accordingly one or more regions can each have an area that is no largerthan about 25 nm², no larger than about 10 nm², no larger than about 5nm², or no larger than about 1 nm². Larger regions are also useful, forexample, to accommodate populations of nucleic acids of various sizes.Thus, one or more regions can each have an area that is no larger thanabout 1 mm², no larger than about 500 μm², no larger than about 100 μm²,no larger than about 25 μm², no larger than about 10 μm², no larger thanabout 5 μm², no larger than about 1 μm², no larger than about 500 nm²,or no larger than about 100 nm². Although there need not be a lowerlimit to the area of an individual region, in particular embodiments theregion will be no smaller than about 1 mm², no smaller than about 500μm², no smaller than about 100 μm², no smaller than about 25 μm² nosmaller than about 10 μm², no smaller than about 5 μm², no smaller thanabout 1 μm², no smaller than about 500 nm², no smaller than about 100nm², no smaller than about 25 nm², no smaller than about 10 nm², nosmaller than about 5 nm², or no smaller than about 1 nm². Indeed, aregion can have a size that is in a range between an upper and lowerlimit selected from those exemplified above. Although several sizeranges for regions of a surface have been exemplified with respect tonucleic acids and on the scale of nucleic acids, it will be understoodthat regions in these size ranges can be used for applications that donot include nucleic acids. It will be further understood that the sizeof the regions need not be confined to a scale used for nucleic acidapplications.

For embodiments that include a plurality of regions, such as an array ofregions, the regions can be discrete, being separated with spacesbetween each other. The size of the regions and/or spacing between theregions can vary such that arrays can be high density, medium density orlower density. High density arrays are characterized as having regionsseparated by less than about 15 μm. Medium density arrays have regionsseparated by about 15 to 30 μm, while low density arrays have regionsseparated by greater than 30 μm. An array useful in the invention canhave regions that are separated by less than 100 μm, 50 μm, 10 μm, 5 μm,1 μm or 0.5 μm.

A metal layer can be deposited on a surface using methods known in theart such as wet plasma etching, dry plasma etching, atomic layerdeposition, ion beam etching, chemical vapor deposition, vacuumsputtering or the like. Any of a variety of commercial instruments canbe used as appropriate including, for example, the FlexAL®, OpAL®,Ionfab 300Plus®, or Optofab 3000® systems (Oxford Instruments, UK). Ametal layer can also be deposited by e-beam evaporation or sputtering asset forth in Thornton, Ann. Rev. Mater. Sci. 7:239-60 (1977), which isincorporated herein by reference in its entirety. Metal layer depositiontechniques, such as those exemplified above, can be combined withphotolithography techniques to create metal regions or patches on asurface. Exemplary methods for combining metal layer depositiontechniques and photolithography techniques are provided in Example Ibelow.

In particular embodiments, a polymerizable material is used. Forexample, a polymerizable material can be contacted with a surface suchas a surface having metal regions, mask regions and/or interstitialregions. Typically polymerizable material is provided in a liquid stateand subsequently converted to a gel, polymer or other semisolidmaterial. Examples of polymerizable materials include, withoutlimitation, acrylamide, methacrylamide, hydroxyethyl methacrylate,N-vinyl pyrrolidinone or derivatives thereof. Such materials are usefulfor preparing hydrogels. In some embodiments, the polymerizable materialcan include two or more different species of compound that form aco-polymer. For example, two or more different species of acrylamide,methacrylamide, hydroxyethyl methacrylate, N-vinyl pyrrolidinone orderivatives thereof can function as co-monomers that polymerize to forma copolymer hydrogel. Exemplary hydrogels and polymerizable materialsthat can be used to form hydrogels are described, for example, in USPat. Pub. No. 2011/0059865 A1, which is incorporated herein by referencein its entirety. Other hydrogels include but are not limited to,polyacrylamide polymers formed from acrylamide and an acrylic acid or anacrylic acid containing a vinyl group as described, for example, in WO00/31148 (incorporated herein by reference in its entirety);polyacrylamide polymers formed from monomers that form [2+2]photo-cycloaddition reactions, for example, as described in WO 01/01143or WO 03/014392 (each of which is incorporated herein by reference inits entirety); or polyacrylamide copolymers described in U.S. Pat. No.6,465,178, WO 01/62982 or WO 00/53812 (each of which is incorporatedherein by reference in its entirety). Silane-free acrylamide (SFA)polymer formed by polymerization of silane free acrylamide and N—(Sbromoacetamidylpentyl)acrylamide (BRAPA) is particularly useful. Methodsfor making and using SFA polymer are set forth in the Examples below.

Other useful polymerizable materials are those that undergo atemperature dependent change in state from liquid to gelatinous.Examples include, but are not limited to agar, agarose, or gelatin.

A polymerizable material can be contacted with a surface usingtechniques convenient to the material and to the surface. Typically thepolymerizable material is in a liquid state and can be manipulated usingknown fluidics techniques. As such, liquids can be moved by hydraulicforces, pneumatic forces, displacement, pumping, gravity flow or thelike. Generally, a liquid is delivered to a surface when performing amethod as set forth herein. However, the surface can be brought to aliquid, for example, by dipping, immersing, floating or the like. Thecontacting of a liquid and a surface can be carried out by automatedmethods or manually. For example, an individual can manually deliver aliquid to a surface using a hand held pipette or the individual canhandle a substrate to bring it into contact with a liquid. Examples ofautomated methods include, but are not limited to, robotic manipulationsto pipette liquids to a surface or robotic manipulations to bring asubstrate into contact with a liquid.

Generally, a liquid, such as a liquid containing a polymerizablematerial, is contacted with a surface in a way to wet the entiresurface. For example, the liquid can form a continuous layer over thesurface. The liquid when in a continuous layer over the surface can haveuniform thickness or depth. For example, the surface can be maintainedin a level orientation with respect to gravity such that the depth ofthe liquid over the surface is uniform. Alternatively, the depth orthickness of the liquid in the continuous layer can differ across thesurface. For example, the surface can be tilted to a non-levelorientation with respect to gravity such that the liquid has a greaterdepth over one region of the surface compared to another region of thesurface. Alternatively or additionally, the surface can contain relieffeatures that create regions of different depth.

In particular embodiments, the liquid can be in discontinuous contactwith one or more regions of a surface (e.g. metal regions, interstitialregions, mask regions, or transparent regions). For example, the liquidcan form droplets on the surface. The surface can contain features thatretain liquids in particular regions and prevent the liquid fromcontacting other regions. For example the surface can include wells orchannels that contain a liquid. Alternatively or additionally, thesurface can have raised regions that are above the surface of a liquidand therefore not in contact with the liquid. Another way to limitcontact of a liquid to only a portion of a surface is to use regionshaving differing hydrophilicity and hydrophobicity. An aqueous liquidcan be retained in a hydrophilic region while being excluded or repelledfrom a hydrophobic region. Alternatively, an organic or non-polar liquidcan be retained in a hydrophobic region while being excluded or repelledfrom a hydrophilic region. Thus, regions set forth herein with respectto particular characteristics can have a further characteristic of beinghydrophobic or hydrophilic.

In embodiments utilizing a flow-cell, a polymerizable material can bedelivered to the flow-cell in a volume that fills the flow-cellpartially or fully. Thus, the flow-cell can contain no gas bubbles (e.g.when fully filled), or it can contain one or more gas bubbles (e.g. whenpartially filled).

A polymerizable material can be kept in a liquid state during one ormore manipulation or step in a method set forth herein. For example, achemically polymerizable material can be kept separated from a secondmaterial or chemical that causes polymerization to form a gel. In afurther example, a photo-polymerizable material can be maintained in adark environment or at least in an environment that is masked, filteredor otherwise blocked from light having a wavelength that induces thephoto-polymerization. The material can also be maintained at atemperature that prevents or inhibits polymerization. For example,agarose can be maintained at a temperature that is above its meltingtemperature or hydrogel precursors can be kept at a temperature thatprevents or inhibits polymerization.

A polymerizable material, once present at a desired location, such as ona surface, can be polymerized using a method appropriate to the materialto form a gel. The appropriate method will be known to those skilled inthe art in view of the known or determinable properties andcharacteristics of the material. For example, a hydrogel can bepolymerized using methods set forth in US Pat. Pub. No. 2011/0059865 A1,WO 00/31148, WO 01/01143, WO 03/014392, U.S. Pat. No. 6,465,178, WO01/62982 or WO 00/53812, each of which is incorporated herein byreference in its entirety. Agar, agarose, gelatin and other materialsthat undergo a temperature dependent solidification can be polymerizedby reducing the temperature of the material to form a gel.

In many embodiments, a gel made or used in a method set forth hereinwill be a continuous gel layer with respect to coverage of a surface.The gel can thus form a layer that is uninterrupted across severalregions of a surface including, for example, two or more metal regionsor interstitial regions on a metal patterned surface. The gel when in acontinuous layer over a surface can have uniform thickness or depth.Alternatively, the depth or thickness of the gel in the continuous layercan differ across the surface. As such the gel is understood to becontinuous with respect to spanning across several regions of a surfaceeven if the thickness of the gel differs over two or more regions of thesurface, so long as the thickness is greater than zero. An example of asystem where a gel can be continuous and yet have different thicknessover different regions is when a gel covers a surface that containsrelief features. Here the relief features create regions of differentdepth, much like the difference in ocean depth over trenches and reefsthat form relief features on the ocean floor. Alternatively oradditionally, the gel can have a greater depth over a region at or neara first end of the surface compared to the depth at a region at or nearthe other end of the surface. Such a difference in depth would occur forexample if the gel formed while the first end of the surface was held ata lower position with respect to gravity (compared to the position ofthe second end).

In particular embodiments, a gel can be discontinuous with respect toits coverage of two or more regions of a surface. More specifically, twoor more regions of gel can be separated by a region where there is nogel. For example, the gel can form two or more discrete features on thesurface. The features can form patches of gel with a surface contactarea (i.e. a footprint) having shapes, sizes or pitch similar to thoseset forth herein with regard to metal patches. Indeed, in someembodiments the gel patch can coat a metal region or interstitial regionsuch that the footprint of the gel patch has perimeter boundariesdefined by the shape and size of the metal region or interstitialregion, respectively. Such gel features can be formed, for example, bythe use of near-field masks and photo-polymerization as set forth inExamples II and III, below. Patches or regions of gel at two or morediscontinuous regions can have the same or different thickness.

The depth of a gel layer, or portion thereof, can differ for particularapplications of the methods set forth herein. The depth can be, forexample, in the nanometer, micron or millimeter range or higher ifdesired. In particular embodiments, a gel layer can have a depth that isat least about 10 nm, 25 nm, 50 nm, 100 nm, 500 nm, 1 μm, 10 μm, 50 μm,100 μm, 500 μm, 1 mm, 10 mm, 100 mm or higher. Alternatively oradditionally, the depth of a gel layer can be at most about 100 mm, 10mm, 1 mm, 500 μm, 100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 100 nm, 50 nm, 25nm, 10 nm or 1 nm. It will be understood that a gel layer made or usedin a method set forth herein can have a depth that ranges between two ofthe exemplary boundaries set forth above. It will also be understoodthat the ranges are merely exemplary and are not intended to limit allembodiments of the invention; rather a gel layer can have a depth thatextends beyond the lower or upper boundaries exemplified above.

A gel, whether continuous or discontinuous, can have non-uniformthickness across different regions of a surface. For example, inembodiments set forth herein, portions of a gel layer that coat metalregions can be thicker than portions of the gel layer that coatinterstitial regions. Such a configuration can result for example, dueto metal-enhanced polymerization of SFA. Thus, a metal patterned surfacecan have gel regions of greater mass over metal patches compared to themass of the gel regions over interstitial regions. The greater mass canbe manifested as one or more of increased thickness, increased densityor increased volume of gel. Exemplary methods believed to form greatergel mass over metal regions compared to interstitial regions areprovided in Example I. Alternatively, portions of a gel layer that coatmetal regions can have lower mass than portions of the gel layer thatcoat interstitial regions. Examples II and III set forth embodimentswherein a lower mass of gel is believed to form over metal regionscompared to the thickness of the gel formed over interstitial regions.The difference in gel mass can correlate with different properties of asurface, such as presence or absence of a metal coating as demonstratedin the Examples, however gel mass can also differ for regions of asurface that are the same. For example, gel thickness, density or volumecan be non-uniform across a metal coating on a surface.

For embodiments where a gel has different mass (e.g. depth, thickness,volume or density) at different locations on a surface, the differencecan range from a difference of a few percentage points to a differenceof several fold. For example, the gel present at a first region of asurface can have at least about 10%, 25%, 50%, 75% or 100% more massthan the gel present at a second comparably sized region of the surface.Larger differences in mass of at least about 2 fold, 3 fold, 4 fold, 5fold, 10 fold, 25 fold 50 fold, 100 fold or more are also useful.Alternatively or additionally, the difference in mass for gel overdifferent, comparably sized regions of a surface can have an upper limitthat is at most about 100 fold, 50 fold, 25 fold, 10 fold, 5 fold, 4fold, 3 fold, 2 fold, 100%, 75%, 50%, 25%, 10% or lower. For example,the difference in mass for a gel over a surface, in many embodiments,can be negligible, or indistinguishable to effectively approach a statewhere the mass of the gel is uniform across a gel layer whether thelayer is continuous or discontinuous over a surface.

A gel can have uniform or non-uniform density across different regionsof a surface. For example, in embodiments set forth herein, portions ofa gel layer that coat metal regions can have higher density thanportions of the gel layer that coat interstitial regions. Higher densitycan be characterized, in some embodiments, as a decreased porosity forthe gel or as an increase in the number of cross links per volume of thegel. Higher density gel can result for example, due to metal inducedpolymerization of SFA. Thus, a metal patterned surface can have higherdensity gel regions over metal patches compared to the density of thegel regions over interstitial regions. Example I provides methodsbelieved to form a higher density gel layer over metal regions comparedto the density of the gel formed over interstitial regions.Alternatively, portions of a gel layer that coat metal regions can beless dense than portions of the gel layer that coat interstitialregions. Examples II and III set forth embodiments wherein a lowerdensity gel layer is believed to form over metal regions compared toover interstitial regions. The difference in gel density can correlatewith different properties of a surface, such as presence or absence of ametal coating as demonstrated in the Examples, however gel density canalso differ for regions of a surface that are the same. For example, geldensity can be non-uniform across a metal coating on a surface.

The formation of a gel layer in the methods set forth herein isexemplary of other molecular layers that can be formed using similarmethods. Accordingly, the methods set forth herein can be used toproduce any of a variety of molecular layers including those that maynot be characterized as gels such as polymer brush surfaces,nanofilament surfaces, etc. Furthermore an array of the presentdisclosure can include a continuous molecular layer in place of thecontinuous gel layers exemplified herein. In particular embodiments, amethod of making a nucleic acid array is provided. The method caninclude the steps of (a) providing a surface having metal regions andinterstitial regions, the interstitial regions having a composition thatdiffers from the metal regions, wherein a continuous gel layer coats thesurface across the metal regions and the interstitial regions; and (b)contacting the continuous gel layer with a fluid including nucleic acidsunder conditions wherein the nucleic acids become attached to the gellayer and wherein a greater amount of the nucleic acids from the fluidattach to portions of the gel layer that coat the metal regions than theamount of nucleic acids that attach to portions of the gel layer thatcoat the interstitial regions.

A method set forth herein, can include a step of attaching a nucleicacid to a gel layer. This can be achieved, for example, by contactingthe gel with a fluid containing the nucleic acids under conditionswherein the nucleic acids attach to the gel. The contacting of thenucleic acid and gel can occur under conditions wherein a uniformconcentration of the nucleic acids contacts the portions of a continuousgel layer that coat different regions of the surface, such as metalregions and interstitial regions. A liquid that contains nucleic acidscan be delivered using methods set forth above in regard to delivery ofliquids bearing polymerizable materials.

Generally covalent attachment of the nucleic acids to the gel isdesired. However, non-covalent attachment can also be useful. Methodsfor attaching nucleic acids to gels are known in the art and include,for example, one or more of those described in US Pat. Pub. No.2011/0059865 A1, WO 2007/135368, or WO 2008/093098, each of which isincorporated herein by reference in its entirety. The nucleic acids canbe attached to the gel via their 3′ oxygen, 5′ oxygen, or at otherlocations along their length such as via a base moiety of the 3′terminal nucleotide, a base moiety of the 5′ nucleotide, and/or one ormore base moieties elsewhere in the molecule. Non-covalent modes ofattachment include, for example, ionic interactions between nucleic acidand gel, entrapment of nucleic acid within pores of a gel, bindingbetween receptors and ligands on the gel and/or nucleic acid, and otherknown modes.

Nucleic acids that are made or used in methods set forth herein can beDNA or RNA or derivatives thereof such as peptide nucleic acids.Exemplary derivatives of nucleic acids and nucleotides that are usefulin the methods and compositions set forth herein are described, forexample, in U.S. Pat. No. 7,582,420; 6,890,741; 6,913,884 or 6,355,431,each of which is incorporated herein by reference in its entirety. Inparticular embodiments a population of nucleic acids having the samesequence is attached to a gel. Thus, the gel can have a single speciesof attached nucleic acids. Alternatively, a population of nucleic acidshaving different sequences can be attached to a gel. For example, apopulation of 2 primer types, useful for amplification of a sequenceflanked by priming sites that complement the 2 primer types, can beattached to a gel. In particular embodiments, different nucleic acids,such as one or more different primer types (i.e. having one or moredifferent sequences) can be distributed randomly throughout the gel. Forexample, a solution of different nucleic acids can be contacted with thegel such that the nucleic acids diffuse to random locations where theycan attach. Alternatively, the different nucleic acids can be located atpredefined or known locations, for example using array techniques.

Exemplary array techniques that are useful include, without limitation,those used in making a Sentrix® Array or Sentrix® BeadChip Arrayavailable from Illumina, Inc. (San Diego, Calif.) or others includingbeads such as those described previously herein. Further arraytechniques are those used in the commercial manufacture of arrays fromAffymetrix such as the GeneChip® microarrays or other microarraysynthesized in accordance with techniques sometimes referred to asVLSIPS™ (Very Large Scale Immobilized Polymer Synthesis) technologies asdescribed, for example, previously herein. Spotting techniques can alsobe used such as those used for manufacture of a CodeLink™ Arrayavailable from Amersham Biosciences. Printing methods are also usefulsuch as those used for SurePrint™ arrays available from AgilentTechnologies.

Accordingly, this disclosure provides a method of making a nucleic acidarray. In particular embodiments, the method includes the steps of (a)providing a surface having metal regions and interstitial regions, theinterstitial regions having a composition that differs from the metalregions, wherein a continuous gel layer coats the surface across themetal regions and the interstitial regions; (b) contacting thecontinuous gel layer with a fluid comprising nucleic acids; and (c)selectively modifying the portions of the gel layer that coat theinterstitial regions compared to the portions of the gel layer that coatthe metal regions, thereby attaching the nucleic acids to the gel layer,wherein a greater amount of the nucleic acids from the fluid attach toportions of the gel layer that coat the metal regions than the amount ofnucleic acids that attach to portions of the gel layer that coat theinterstitial regions.

Nucleic acids that are attached to a gel, for example in the format ofan array but also in other formats, can be used for any of a variety ofpurposes. A particularly desirable use for the nucleic acids is to serveas capture probes that hybridize to target nucleic acids havingcomplementary sequences. The target nucleic acids once hybridized to thecapture probes can be detected, for example, via a label recruited tothe capture probe. Methods for detection of target nucleic acids viahybridization to capture probes are known in the art and include, forexample, those described in U.S. Pat. No. 7,582,420; 6,890,741;6,913,884 or 6,355,431 or US Pat. Pub. Nos. 2005/0053980 A1;2009/0186349 A1 or US 2005/0181440 A1, each of which is incorporatedherein by reference in its entirety. For example, a label can berecruited to a capture probe by virtue of hybridization of the captureprobe to a target probe that bears the label. In another example, alabel can be recruited to a capture probe by hybridizing a target probeto the capture probe such that the capture probe can be extended byligation to a labeled oligonucleotide (e.g. via ligase activity) or byaddition of a labeled nucleotide (e.g. via polymerase activity).

A further use for nucleic acids that are attached to a gel is forpriming amplification of a template nucleic acid. In an exemplarymethod, the template nucleic acid hybridizes to a gel-attached primerand the 3′ end of the primer is extended to create a complementary copyof the template. In some embodiments two different primers can beattached to the gel. The primers can form a pair used for amplificationof a template and its complementary copy. As such, two primers can beused for amplification of the template into multiple copies to form acluster or population of amplicons. For example, amplification can becarried out using bridge amplification to form nucleic acid clustersattached to the gel. Useful bridge amplification methods are described,for example, in U.S. Pat. Nos. 5,641,658 and 7,115,400; U.S. Pat. Pub.Nos. 2002/0055100 A1, 2004/0096853 A1, 2004/0002090 A1, 2007/0128624 A1,and 2008/0009420 A1, each of which is incorporated herein by referencein its entirety. Another useful method for amplifying nucleic acidsusing a gel-attached primer is rolling circle amplification (RCA). RCAcan be carried out, for example, as described in Lizardi et al., Nat.Genet. 19:225-232 (1998) and US Pat. Pub. No. 2007/0099208 A1, each ofwhich is incorporated herein by reference in its entirety. The primerscan also be used in a multiple displacement amplification (MDA)reaction, for example, using a product of RCA (i.e. an RCA amplicon) asa template. Exemplary methods of MDA are described in U.S. Pat. Nos.6,124,120; 5,871,921; and EP 0,868,530 B1, each of which is incorporatedherein by reference in its entirety.

A surface having a metal region and interstitial region can furtherinclude one or more nucleic acids over the metal region. Additionally,one or more nucleic acids can optionally be present over theinterstitial region. As set forth above, the nucleic acids over theseregions are typically attached to a gel. However, a gel is optional andneed not be present for all embodiments of the invention. In someembodiments the amount or concentration of nucleic acid molecules overthe metal region will exceed the amount or concentration of nucleicacids over the interstitial region. For example, the amount orconcentration of nucleic acids over the metal region can be at leastabout 10%, 25%, 50%, 75% or 100% more than the amount or concentrationof nucleic acids present over a comparably sized area of theinterstitial region. Larger differences in amount or concentration of atleast about 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 25 fold 50 fold,100 fold or more are also possible. Alternatively or additionally, thedifference in the amount or concentration of nucleic acids over themetal region in comparison to a comparable sized area of theinterstitial region can have an upper limit that is at most about 100fold, 50 fold, 25 fold, 10 fold, 5 fold, 4 fold, 3 fold, 2 fold, 100%,75%, 50%, 25%, 10% or lower. The amount or concentration of nucleicacids present over a metal region can be between two or more of theexemplary boundaries above. Similar boundaries and ranges can occur forthe amount or concentration of nucleic acids present over other maskregions as well. In particular embodiments, the amount or concentrationof nucleic acids over the interstitial regions is substantially none.

The amount or concentration of nucleic acid molecules over a metalregion can exceed the amount or concentration of nucleic acids over aninterstitial region in embodiments wherein the metal region producesincreased density of gel or produces increased grafting of primers tothe gel. The methods set forth in Example I can be used for productionof a patterned surface wherein a greater amount or concentration ofnucleic acid molecules is present over metal regions compared tointerstitial regions. Selective photolysis or laser ablation of nucleicacids over interstitial regions, while masking nucleic acids over metalregions can also be used. An exemplary method utilizing masking andlaser ablation is described in Example II. Mask regions havingnon-metallic materials can be used similarly.

In an alternative method, masking can be used to selectively prevent orreduce attachment of nucleic acids over metal regions (or regions ofother mask material) compared to over interstitial regions. Again thenucleic acids over the two types of regions can optionally be attachedto a gel layer. In one example, a photo-chemically activatedcross-linking agent can be used to selectively polymerize apolymerizable material to form a gel over an interstitial region,whereas polymerizable material over a metal region is masked fromphoto-chemical activation such that little to no gel forms over themetal region. An exemplary technique is set forth in Example III.Similarly, a photo-chemically active agent can be used to graft anucleic acid to a gel such that irradiation with the proper wavelengthresults in grafting of the nucleic acid to a gel or to the surface overthe interstitial region, whereas little to no nucleic acid is grafted tothe gel or surface over the metal region.

A further exemplary method for selectively photo-polymerizing materialto form a gel over an interstitial region is shown in FIG. 10 andfurther described in the provisional application U.S. Ser. No.61/657,508. As shown in the figure, acrylamide and a new monomer 4c(synthesized via a simple one-step procedure), can be polymerized usingfree-radical polymerization analogous to the preparation ofpolyacrylamide.

Accordingly, the amount or concentration of nucleic acid molecules overan interstitial region can exceed the amount or concentration of nucleicacids over a comparably sized area of a metal region or other maskregion. For example, the amount or concentration of nucleic acids overthe interstitial region can be at least about 10%, 25%, 50%, 75% or 100%more than the amount or concentration of nucleic acids present over themetal region or other mask region. Larger differences in amount orconcentration of at least about 2 fold, 3 fold, 4 fold, 5 fold, 10 fold,25 fold 50 fold, 100 fold or more are also possible. Alternatively oradditionally, the difference in the amount or concentration of nucleicacids over the interstitial region in comparison to a comparably sizedarea of a metal region (or mask region) can have an upper limit that isat most about 100 fold, 50 fold, 25 fold, 10 fold, 5 fold, 4 fold, 3fold, 2 fold, 100%, 75%, 50%, 25%, 10% or lower. The amount orconcentration of nucleic acids present over an interstitial region canbe between two or more of the exemplary boundaries above. In particularembodiments, the amount or concentration of nucleic acids over the metalregions is substantially none.

Although methods for making and using surfaces are exemplified hereinwith respect to nucleic acids, it will be understood that other analytescan be attached to a gel. Exemplary analytes include, withoutlimitation, nucleotides, amino acids, proteins, peptides, antibodies,lectins, sugars, polysaccharides, metabolites, candidate compounds of asynthetic library and analogs thereof. Known methods can be used tosynthesize, attach and detect any of a variety of analytes used in amethod set forth herein.

A metal region can optionally be removed from a surface. For example, ametal coating can be removed from a surface by physical or chemicalmeans. Exemplary physical means include, but are not limited to,polishing, sanding, lapping or grinding the surface to remove the metalcoat with abrasive forces; heating the surface to thermally melt ordegrade the metal; peeling the metal coating from the surface; laserablating the metal from the surface, or the like. Exemplary chemicalmeans include, without limitation, treatment with acid, base (i.e.alkaline treatment), caustic solvents, Al₂O₃ hydrolysis, or the like.Specific examples of useful etchants include, without limitationpersulfate, sulfuric acid, chromic-sulfuric acid, orthophosphoric acidand derivatives thereof such as TRANSETCH-N (Transene Company Inc.,Danvers Mass.), cupric chloride with the copper complexed with ammonia,and etchants described in Williams et al., J. MicromelectromechanicalSys. 12:761-778 (2003) (incorporated herein by reference in itsentirety) such as isotropic silicon etchant, potassium hydroxide (KOH),hydrofluoric acid (HF), buffered hydrofluoric acid (BHF), hot phosphoricacid, titanium wet etchant, chromium etchant, molybdenum etchant, warmhydrogen peroxide, copper etchants, hot sulfuric and phosphoric acids,acetone, methanol, isopropanol, xenon difluoride, HF and H₂O vapor, andoxygen plasma. A combination of physical and chemical means can be usedto remove a metal coating from a surface.

Removal of a metal region from a surface can be carried out at differentpoints in the processing of a substrate in accordance with a desired usefor the substrate. For example, a metal coating can be removed before orafter a gel is formed on the surface that is coated by the metal. In thelatter case, the metal coating can be removed using conditions thatpreserve the gel layer on the surface. In particular embodiments, themetal coating can be removed after formation of the gel and aftergrafting of nucleic acids to the gel. Furthermore, if desired the metalcoating can be removed after formation of the gel, after grafting ofnucleic acids to the gel and after the nucleic acids are used in aprocess such as hybridization to other nucleic acids, bridgeamplification, detection of labels present at the nucleic acids or atamplicons produced from the nucleic acids, or a combination of these orsimilar steps carried out on the nucleic acids grafted to the gel. Asset forth in Example I, metal can be removed from a surface before,after or during removal of a photoresist material from the surface.

A variety of manipulations that can be carried out during themanufacture and use of a surface are set forth in further detail herein,or known in the art, one or more of which can be carried out prior toremoval of a metal from the surface. Thus, the metal regions can beremoved from the surface under conditions wherein the nucleic acidsremain attached to the gel layer on the surface. Alternatively, a metalregion can be removed from a surface prior to carrying out one or moreof the manipulations or steps set forth above or elsewhere herein.

Retaining a metal coating on a surface can provide advantages for makingand using the surface. For example, a metal region can provide anear-field mask to protect regions of a liquid, gel or chemicalcomponents attached to the gel (such as nucleic acids, proteins, andother analytes of interest) from radiation. This protection can blocksubstances located opposite the mask (the orientation being with respectto impinging radiation) from the effects of photolysis, photo-cleavage,photo-ablation, photo-chemical modification and other photo-chemicalprocesses. For embodiments that use a metal patterned surface, regionsof a liquid, gel or chemical components attached to the gel that coat orare otherwise present at non-metal containing, interstitial regions willnot be masked from the radiation and can therefore experience theeffects of photo-chemical processes. Examples II and III set forthmethods where metal regions are used as near-field masks. Retaining ametal coating can also provide advantages for the detection of analytes.For example, the metal surface can serve to reflect optical signals toachieve signal amplification during fluorescence detection methods orother optical detection methods. Other advantages will be apparent tothose skilled in the art in view of the characteristics of the metalcoated surface as set forth herein and further in view of the desireduse of the surface.

The use of near-field masks on surfaces and photo-induced manipulationsof materials on the surfaces is often exemplified herein with respect tometal regions, glass or silica interstitial regions and radiation havingwavelengths in the UV-VIS-IR parts of the radiation spectrum. However,it will be understood that the methods are also applicable to other maskmaterials, interstitial region materials and radiation wavelengths.

Accordingly methods are provided for preparing a surface, including thesteps of (a) providing a surface having mask regions and transparentregions, the mask regions having a composition that blocks transmittanceof radiation in a first part of the electromagnetic radiation spectrumand transparent regions having a composition that transmits radiation inthe first part of the electromagnetic radiation spectrum; (b) contactingthe surface with a fluid comprising a photo-polymerizable material,wherein the fluid coats the surface across the mask regions and thetransparent regions; and (c) selectively photo-polymerizing the portionsof the fluid that coat the transparent regions over the portions of thefluid that coat the mask regions, thereby forming a gel that coats thetransparent regions.

Also provided is a method of making a nucleic acid array. The method caninclude the steps of (a) providing a surface having mask regions andtransparent regions, the mask regions having a composition that blockstransmittance of radiation in a first part of the electromagneticspectrum and transparent regions having a composition that transmitsradiation in the first part of the electromagnetic radiation spectrum,wherein a continuous gel layer coats the surface across the mask regionsand the transparent regions; (b) contacting the continuous gel layerwith a fluid including nucleic acids under conditions wherein thenucleic acids become attached to the gel layer and wherein a firstsubpopulation of the nucleic acids attach to portions of the gel layerthat coat the mask regions and a second subpopulation of nucleic acidsattach to portions of the gel layer that coat the transparent regions;and (c) irradiating the surface with radiation in the first part of theelectromagnetic spectrum, thereby selectively modifying the nucleicacids of one of the subpopulations compared to the nucleic acids of theother subpopulation.

As set forth previously herein, a mask region can be made from one ormore metal materials. Other materials are also useful when used to maskradiation of an appropriate wavelength. For example, glass can serve asa mask for radiation in the far UV. Glass can be doped with dyes orother materials that are opaque to radiation in a particular wavelength.Band gap semiconductors are also useful. Exemplary materials that can beused for a mask include, but are not limited to those, used incommercial optical filters available from suppliers such as EdmundOptics (Barrington N.J.), Chroma (Bellows Falls, Vt.), and Omega Optical(Brattleboro, Vt.). The use of such materials can provide masking indesired wavelength ranges, including for example, in one or more of theUV, VIS, or IR parts of the radiation spectrum. The mask can provide acutoff such that radiation below a certain wavelength is blocked or suchthat radiation above a certain wavelength is blocked. In certainembodiments the mask can act as a bandpass filter to pass wavelengthswithin a particular range while blocking wavelengths outside of thisrange. Alternatively, the mask can act as a bandstop filter to blockwavelengths within a particular range while passing wavelengths outsideof this range.

Radiation used in a method set forth herein can be derived from any of avariety of sources that is appropriate for generation of radiation in adesired wavelength range. Exemplary sources include a lamp such as anarc lamp or quartz halogen lamp; light emitting diode (LED); or laser,such as a solid state laser, dye laser, photonic crystal laser,semiconductor laser or gas laser. Accordingly, the radiation can becollimated as produced by a laser or non-collimated as produced from alamp. The radiation can be directed to a surface such that it impingeson the side of a mask that is opposite the side where a material to beprotected resides. For example, radiation can be directed to theunderside of a flow-cell or other substrate, such that it hits theunderside of a mask whereby a liquid, gel or nucleic acid present overthe mask (e.g. as a coating) is protected from the radiation. Anyliquid, gel or nucleic acid present at a location over a transparentregion of the flow-cell or substrate will be contacted with theradiation in this configuration.

Radiation that passes through a surface can selectively modify amaterial coating the surface, or otherwise present over the surface, inany of a variety of ways. As set forth above, the modification can beselective with respect to being greater for material above a transparentregion of the surface as compared to material above a mask region. Forexample, material above the mask region can be substantially unmodifiedwhile material above the transparent region is modified. The degree ofmodification can be controlled for example by duration of irradiation,intensity of radiation or a combination thereof. As such the amount of amaterial above a mask region of a surface that is modified can be lessthan about 50%, less than about 25%, less than about 10%, less thanabout 5% or less than about 1% of the amount that is modified above acomparably sized area of a transparent region of the surface.

Any of a variety of the materials set forth herein as coating orotherwise being present over a transparent region of a surface can bemodified by radiation in a method or composition set forth herein. Forexample, a polymerizable material can be modified to form a gel layervia photo-activation of a photo-reactive crosslinking group. In anotherexample, a gel material can be heated, ablated, photolysed, or modifiedby attachment of a moiety from a photo-reactive reagent. Exemplaryphoto-reactive reagents are photo-crosslinkers that form a linkerbetween a gel and another moiety (e.g. a moiety can be a surface ornucleic acid) when irradiated by the appropriate wavelength of light.Other photo-reactive reagents are photo-reactive moieties that arepresent on a gel or on a moiety that is to be attached to a gel. In thiscase irradiation with the appropriate wavelength of light will cause alinkage between the gel and the moiety. Photo-reactive reagents areknown in the art and can be obtained from commercial sources such asInvitrogen (a subsidiary of Life Technologies, Carlsbad, Calif.), GlennResearch (Sterling, Va.), Thermo Scientific (Rockford, Ill.) orSigma-Aldrich (St. Louis, Mo.). Exemplary moieties that can be attachedto a gel by photo-reaction are a nucleic acid, protein, antibody,metabolite, polysaccharide, nucleotide, amino acid or other analyte ofinterest, such as those set forth elsewhere herein.

Analytes, such as nucleic acids, can be modified in other ways byradiation as well. For example the photo-reactive reagents set forthabove can be used to attach the nucleic acids to the surface of asubstrate, to attach two or more nucleic acids to each other, or toattach another analyte to the nucleic acid. Other radiation mediatedmodifications of nucleic acids include photolysis, ablation, removal, orbleaching to destroy an optical label present on the nucleic acid. Forexample, a nucleic acid can be attached to a gel or surface via aphoto-cleavable linker such that irradiation releases the nucleic acid.The nucleic acid can then be washed away. A particularly usefulphoto-reaction is crosslinking of nucleic acids strands using psoralenirradiated with UV light.

In some embodiments a flow-cell or other vessel having multiple surfacesis used. Vessels having multiple surfaces can be used such that only asingle surface is treated using methods set forth herein. Alternativelytwo or more surfaces present in the vessel can be treated. For example,opposing surfaces in the interior of a flow cell can be selectivelyaddressed with focused radiation using methods known in the art such asconfocal techniques. Useful confocal techniques and devices forselectively directing radiation to multiple surfaces of a vessel (e.g. aflow cell) are described, for example, in US 2009/0272914 A1, which isincorporated herein by reference in its entirety.

Alternatively or additionally to the use of focusing techniques,different surfaces of a vessel, such as opposing interior surfaces of aflow cell, can be selectively addressed by masking with a masking liquidor masking gel. For example, a liquid that is opaque to UV radiation canbe introduced into a flow cell prior to treating a first interiorsurface (e.g. the bottom interior surface) of the flow cell with UVlight. At least portions of the first interior surface of the flow cellcan be transparent to the UV light such that the portions will bealtered by the UV light, for example, to photo-cleave a gel or analyte,or to photo-activate a gel or analyte in accordance with techniques setforth herein. However, UV light will be prevented from contacting theopposing interior surface (e.g. the top interior surface) of the flowcell since the UV light will be masked by the liquid that is present inthe flow cell. The use of a masking liquid or masking gel that is opaqueto radiation of a particular wavelength is particularly useful when twoor more surfaces are otherwise susceptible to radiation at thatwavelength (e.g. the surfaces may both be modified by the radiation ordetected due to the radiation). The masking liquid or masking gel can beused to selectively mask at least one surface (e.g. a surface that isoriented opposite a target surface with respect to the direction fromwhich radiation impinges). An advantage of using a liquid mask or gelmask in this way is that the mask can optionally be removed from thevessel such that the vessel can be used for subsequent manipulationswhere masking between surfaces is not desired. Of course in someembodiments, the masking liquid or masking gel need not be removed, forexample, if it does not have properties of blocking radiation of asecond wavelength that is subsequently used with the vessel.

Those skilled in the art will know or be able to determine anappropriate masking liquid or masking gel to use based on the guidanceset forth herein, the desired wavelength of radiation to be masked, andknown optical properties of masking liquids or gels. For example,radiation in the UV range can be masked using benzophenone, titaniumdioxide or carbon black. The wavelength regions that can be masked orblocked include, without limitation, those set forth herein with regardto other embodiments. Furthermore, the concentration of masking orabsorbing species in a liquid or gel can be selected to influence thedepth of penetration for a particular wavelength of radiation into theliquid. The depth of penetration can be readily predicted using Beer'sLaw and known optical properties of the masking or absorbing species.Thus, a masking liquid or masking gel can allow treatment of a materialthat is on a vessel or in a vessel at a particular distance from asurface, while masking material that is on the vessel or in the vesselat a location beyond the particular distance.

Furthermore, in the case of embodiments set forth herein that usesurfaces covered with a gel layer, a masking liquid or masking gel canbe used that does not penetrate the gel layer. In such an embodiment,radiation can pass into a vessel through an exterior surface to whichthe gel layer is attached and treatment or detection of the gel layer(or contents therein) can be achieved. In this scenario, a maskingliquid or masking gel that is present outside of the gel layer canprevent the radiation from impinging the volume of the vessel (or othersurfaces of the vessel) that are outside of the gel layer.Alternatively, if desired a masking liquid or masking gel can be usedthat penetrates a gel layer, thereby masking all or part of the gellayer from radiation of a particular wavelength.

This disclosure further provides a method for preparing a surface thatincludes the steps of (a) providing a surface having mask regions andtransparent regions, the mask regions having a composition that blockstransmittance of radiation in a first part of the electromagneticradiation spectrum and transparent regions having a composition thattransmits radiation in the first part of the electromagnetic radiationspectrum, the surface further including photo-reactive crosslinkingreagents attached thereto; (b) contacting the surface with a fluidcomprising a photo-polymerizable material, wherein the fluid coats thesurface across the mask regions and the transparent regions; and (c)selectively irradiating the portions of the fluid that coat thetransparent regions compared to the portions of the fluid that coat themask regions, wherein the portions of the fluid that coat thetransparent regions are photo-polymerized to form a gel and wherein thegel is photo-crosslinked to the surface at the transparent regions.

As exemplified by the above method, a material that is present over atransparent region of a surface (e.g. in a fluid form or gel form) canbe modified by radiation in a method or composition set forth herein.For example, a polymerizable material can be modified to form a gellayer via photo-activation of a photo-reactive crosslinking group and/orthe material can be photo-crosslinked to the surface.

In some embodiments, the photo-reactive crosslinking reagents includeoptionally substituted phenyl azide groups. In some of theseembodiments, the phenyl azide is prepared by reacting an amino groupwith N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HSAB). In someembodiments, the phenyl azide is photo-activated prior to polymerizingthe polymerizable material on the surface of the substrate. In apreferred embodiment of such methods, the photo-activated functionalgroups generate nitrene. In another preferred embodiment of suchmethods, the polymer coating is covalently bonded to nitrene viaphoto-activation.

Sulfo-HSAB is a commercially available bifunctional crosslinking agenthaving a photoactive aryl azide and an activated NHS unit. Upon exposureto UV light (˜254-365 nm), the aryl azide generates a nitrene with therelease of nitrogen. This highly reactive species can undergo a varietyof rapid insertion reactions. A pathway to prepare a photoactive surfaceis shown in FIG. 8 and further described in the provisional applicationU.S. Ser. No. 61/657,508. Briefly, a surface is pre-treated with APTS(methoxy or ethoxy silane) and baked to form an amine monolayer. Theamine groups are then reacted with sulfo-HSAB to form an azidoderivative. UV activation then generates the active nitrene species,which can readily undergo a variety of insertion reactions withpoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM).

A photo-reactive crosslinking reagent, like the azido derivative ofsulfo-HSAB, can be located on a surface and photo-crosslinked to apolymer like PAZAM using UV exposure at transparent areas of a surface.The surface can have both mask regions and interstitial regions suchthat the polymer is selectively linked to interstitial regions and notto mask regions. An exemplary process is demonstrated in FIG. 9. Asshown, the mask region is left free of any coupled polymer because thephoto-reactive crosslinker is activated only (and in some casesinitially located only) at the transparent patch. The radical insertionreaction is confined to a monolayer at the surface minimizing theuncontrolled propagation of radicals. Aryl azides functionalized withsilane and phosphate units can also be readily accessed fromcommercially available starting materials, allowing anchoring to a widevariety of patch types. Further exemplary reagents and methods forphoto-crosslinking a gel to a surface are described in the provisionalapplication U.S. Ser. No. 61/657,508.

This disclosure further provides a nucleic acid array. The array caninclude a surface having metal regions and interstitial regions having acomposition that differs from the metal regions, wherein a continuousgel layer coats the surface across the metal regions and theinterstitial regions, wherein nucleic acids are attached to thecontinuous gel layer, and wherein a greater amount of the nucleic acidsare attached to portions of the gel layer that coat the metal regionsthan the amount of nucleic acids that attach to portions of the gellayer that coat the interstitial region.

In particular embodiments, a nucleic acid array can have a surface with(a) mask regions or metal regions, wherein individual mask regions areattached to a single (i.e. no more than one) nucleic acid template; and(b) interstitial regions, wherein a continuous gel layer coats thesurface across the mask regions and the interstitial regions, wherein aplurality of nucleic acid copies of the template nucleic acid areattached to the continuous gel layer in respective clusters surroundingthe metal regions. The mask regions or metal regions can have acomposition that blocks transmittance of electromagnetic radiation in afirst part of the electromagnetic radiation spectrum and theinterstitial regions can have a composition that transmits radiation inthe first part of the electromagnetic radiation spectrum.

Also provided is a nucleic acid array having a surface with mask regionshaving a composition that blocks transmittance of electromagneticradiation in a first part of the electromagnetic radiation spectrum andtransparent regions having a composition that transmits radiation in thefirst part of the electromagnetic radiation spectrum, wherein acontinuous gel layer coats the surface across the mask regions and thetransparent regions, wherein nucleic acids are attached to thecontinuous gel layer, and wherein a greater amount of the nucleic acidsare attached to portions of the gel layer that coat the transparentregions than the amount of nucleic acids that are attached to portionsof the gel layer that coat the mask regions.

Further provided is a nucleic acid array having a surface with maskregions having a composition that blocks transmittance ofelectromagnetic radiation in a first part of the electromagneticradiation spectrum and transparent regions having a composition thattransmits radiation in the first part of the electromagnetic radiationspectrum, wherein a continuous gel layer coats the surface across themask regions and the transparent regions, wherein nucleic acids areattached to the continuous gel layer, and wherein a greater amount ofthe nucleic acids are attached to portions of the gel layer that coatthe mask regions than the amount of nucleic acids that are attached toportions of the gel layer that coat the transparent regions.

Flow cells provide a convenient format for housing an array that isproduced by the methods of the present disclosure and that is subjectedto a sequencing-by-synthesis (SBS) or other detection technique thatinvolves repeated delivery of reagents in cycles. For example, toinitiate a first SBS cycle, one or more labeled nucleotides, DNApolymerase, etc., can be flowed into/through a flow cell that houses anucleic acid array made by methods set forth herein. Those sites of anarray where primer extension causes a labeled nucleotide to beincorporated can be detected. Optionally, the nucleotides can furtherinclude a reversible termination property that terminates further primerextension once a nucleotide has been added to a primer. For example, anucleotide analog having a reversible terminator moiety can be added toa primer such that subsequent extension cannot occur until a deblockingagent is delivered to remove the moiety. Thus, for embodiments that usereversible termination, a deblocking reagent can be delivered to theflow cell (before or after detection occurs). Washes can be carried outbetween the various delivery steps. The cycle can then be repeated ntimes to extend the primer by n nucleotides, thereby detecting asequence of length n. Exemplary SBS procedures, fluidic systems anddetection platforms that can be readily adapted for use with an arrayproduced by the methods of the present disclosure are described, forexample, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S.Pat. No. 7,057,026; WO 91/06678; WO 07/123,744; U.S. Pat. Nos.7,329,492; 7,211,414; U.S. Pat. Nos. 7,315,019; 7,405,281, and US2008/0108082, each of which is incorporated herein by reference in itsentirety.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and 6,274,320, each of which is incorporated herein by reference in itsentirety). In pyrosequencing, released PPi can be detected by beingimmediately converted to adenosine triphosphate (ATP) by ATPsulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons. Thus, the sequencing reaction can bemonitored via a luminescence detection system. Excitation radiationsources used for fluorescence based detection systems are not necessaryfor pyrosequencing procedures. Useful fluidic systems, detectors andprocedures that can be used for application of pyrosequencing to arraysof the present disclosure are described, for example, in WIPO Pat. App.Ser. No. PCT/US11/57111, US 2005/0191698 A1, U.S. Pat. Nos. 7,595,883,and 7,244,559, each of which is incorporated herein by reference in itsentirety.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference in its entirety. Some embodiments can includesequencing-by-hybridization procedures as described, for example, inBains et al., Journal of Theoretical Biology 135(3), 303-7 (1988);Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.,Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which isincorporated herein by reference in its entirety. In bothsequencing-by-ligation and sequencing-by-hybridization procedures,nucleic acids that are present at sites of an array are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein or in references citedherein can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs).Techniques and reagents for FRET-based sequencing are described, forexample, in Levene et al. Science 299, 682-686 (2003); Lundquist et al.Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci.USA 105, 1176-1181 (2008), the disclosures of which are incorporatedherein by reference in its entirety.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US 2009/0026082 A1; US2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, each ofwhich is incorporated herein by reference in its entirety.

Another useful application for an array of the present disclosure, forexample, having been produced by a method set forth herein, is geneexpression analysis. Gene expression can be detected or quantified usingRNA sequencing techniques, such as those, referred to as digital RNAsequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array or using amultiplex assay, the products of which are detected on an array. Anarray of the present disclosure, for example, having been produced by amethod set forth herein, can also be used to determine genotypes for agenomic DNA sample from one or more individual. Exemplary methods forarray-based expression and genotyping analysis that can be carried outon an array of the present disclosure are described in U.S. Pat. No.7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos.2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which isincorporated herein by reference in its entirety.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Preparation and Analysis of Aluminum Oxide PatternedFlow-cells

This example demonstrates preparation of a glass substrate havingaluminum oxide metal surface regions and interstitial surface regionswhere the metal is not present on the glass surface. This Example alsodemonstrates enhancement of DNA cluster formation on the metal-coatedsurfaces compared to the interstitial surfaces.

Glass flow-cells (Illumina, Inc., San Diego, Calif.) were coated withaluminum oxide patches as shown in FIG. 1A or 1B. Briefly, FIG. 1A showsa profile of a glass flow-cell treated to create metal patches (alsoreferred to as metal regions) using a lift-off approach. Morespecifically, a photoresist layer was evenly coated over the surface ofthe glass flow-cell and patches of the photoresist were removed byphotolithography to expose patches of the glass surface. A layer ofaluminum oxide was then deposited on the surface to form a continuousthin film over the photoresist regions and glass patches. Aluminum oxidewas deposited using e-beam evaporation or sputtering as set forth inThornton, Ann. Rev. Mater. Sci. 7:239-60 (1977), which is incorporatedherein by reference in its entirety. The photoresist layer was thenremoved by Acetone lift off to leave aluminum oxide patches. In thelift-off approach the transparent regions of the photomask used forphotolithography corresponded to regions of the flow cell that wouldeventually become metal patches, whereas the mask regions of thephotomask corresponded to regions of the flow cell that would eventuallybecome interstitial regions that separate metal patches.

FIG. 1B shows a profile of a glass flow-cell treated to create metalpatches using an etching approach. An aluminum oxide layer was depositedto create a metal layer on the glass surface of the flow cell, againusing e-beam evaporation or sputtering. A photoresist layer was thencoated over the metal layer and photolithography was carried out using aphotomask to produce patches of photoresist on the metal surface. Theflow cell was then treated with sodium hydroxide to etch metal that wasnot protected by the photoresist patches. The photoresist patches wereremoved with acetone leaving metal patches corresponding to regions ofthe metal surface that had been protected from etching by thephotoresist patches. The metal patches were separated by interstitialregions where metal had been removed to expose the glass surface. In theetching approach the mask regions of the photomask used forphotolithography corresponded to regions of the flow cell that wouldeventually become metal patches, whereas the transparent regions of thephotomask corresponded to regions of the flow cell that would eventuallybecome interstitial regions that separate metal patches.

The aluminum oxide patches that resulted from the above treatments were5 to 50 nm thick, circular in shape with a diameter of about 250 nm, andsurrounded by interstitial regions of glass surface such that the metalregions were separated by a pitch of 750 nm. A diagrammaticrepresentation of the surface is shown in FIG. 2A.

The aluminum oxide-coated flow-cell was then coated with silane-freeacrylamide (SFA) as described at pages 61-62 of WO 2008/093098 (which isincorporated herein in its entirety) except that the temperature for thepolymerization reaction was 35° C., the reaction time was 30-40 min, theamount of TEMED used was 11 μl, and the amount of potassium persulfateused was 96 μl. Primers were grafted to the polymerized SFA as describedin WO 2008/093098. DNA templates were hybridized to the primers andamplified by bridge amplification to form DNA clusters also as describedin WO 2008/093098.

Surprisingly, for flow-cells prepared as described above a 3 to 10 foldincrease in primer density was observed over metal patches as comparedto the density of primers over interstitial regions. Similarly, a 3 to10 fold increase in signal intensity was observed for DNA clustersformed over metal patches compared to signal detected from interstitialregions

A diagrammatic representation of a flow-cell resulting from the abovemethods is shown in FIG. 2B. As shown in the figure an SFA polymer layercoats the flow-cell surface, covering the metal patches and interstitialglass regions as a continuous layer. The primers are attached to the SFAlayer. Although primers are attached to locations throughout the SFApolymer layer, the concentration of primers is substantially higher overthe aluminum oxide, metal patches than over the interstitial glassregions. Template DNA molecules form clusters primarily over thealuminum oxide metal patches, with little to no template attachment oramplification over the interstitial regions.

FIG. 3 shows photographic images and related measurements for flow-cellsprepared generally as set forth above (with exceptions as indicated).FIG. 3A shows a flow-cell substrate, and a magnified view of a portionthereof, after being coated with 3 micron diameter, aluminum oxidepatches. FIG. 3B shows the flow-cell surface after the followingtreatment. The substrate having 3 micron aluminum oxide patches wascoated with SFA and the SFA was then attached to primers as describedabove. The primers were then hybridized to probes having a Texas Red dyeand excess, unhybridized probes were washed away. The hybridized probeswere detected using excitation at 589 nm and emission at 615 nm. Thefluorescence image in the left side panel of FIG. 3B shows a pattern offluorescent signals from primer populations that form over the metalregions. A substantial contrast can be seen between primer patchesformed over metal regions as compared to the relatively low signalproduced from interstitial glass regions. This contrast is indicative ofthe specificity with which the primers attach to SFA at metal regionscompared to SFA at interstitial glass regions. The plot of gray valuesvs. distance in the right side panel of FIG. 3B shows a pattern ofsignals from the primer regions that correlates with the diameter andpitch of the metal regions.

FIGS. 3C and 3D show magnified views of a flow-cell after patterningwith 3 micron diameter aluminum oxide patches, coating with SFA polymer,grafting of primers, and performing bridge amplification to formclusters. The clusters were treated with a SYBR Green labeledintercalating agent, washed to remove non-intercalated agent andfluorescence was detected using a fluorescence microscope. The images inFIGS. 3C and 3D show that clusters formed in a regular pattern that wascorrelated with the pattern of metal patches on the surface. The plot ofgray values vs. distance shows that although some signal was present ininterstitial regions, the signal from clusters formed over metal patcheswas far larger.

Example II Application of Metal Patches as Near-Field Photo-Masks

This Example describes creation of an array of nucleic acid features. Asurface having metal patches separated by interstitial regions wascoated with a gel and photo-cleavable nucleic acid primers were attachedto the gel. Primers that attached to portions of the gel that coatinterstitial regions were photo-cleaved while primers that were attachedto portions of the gel that coated metallic patches were masked fromphoto-cleavage. The remaining patches of nucleic acid primers formed anarray of features on the surface.

Glass flow-cells (Illumina, Inc., San Diego, Calif.) were coated withgold patches using methods set forth in Example I, except that metaldeposition was carried out with gold instead of aluminum oxide. Theresulting flow-cell had eight lanes each with 12 gold patches separatedby glass interstitial regions. The gold-coated flow-cell was then coatedwith silane-free acrylamide (SFA) as described in Example I. Primerswere grafted to the SFA coated flow-cell using the primer graftingmethods described in Example I. The primers in lanes 1-4, 7 and 8 weregrafted to the SFA layer via a nitrobenzyl UV cleavable moiety (GlennResearch, Sterling, Va.). The primers in lanes 5 and 6 did not have aphoto-reactive group and were therefore not UV cleavable. The flow cellis oriented in FIG. 4 such that lanes 1 through 8 are in order from topto bottom. The grafted primers were then hybridized with complementaryprobes having Texas Red labels.

Following hybridization of labeled probes, the flow-cell was then washedand placed over a UV light source. The flow-cell was positioned withrespect to the UV (302 nm) light source such that the gold patchescreated a mask for primers attached over the patches while any primersattached over interstitial regions were exposed to the UV light.Furthermore, a large mask was placed between the UV source and half ofthe flow-cell as shown in FIG. 4A to create a UV exposed area and acontrol area that was not exposed to UV. UV exposure occurred for 2minutes at room temperature. As a result of the experimental set-up, theUV exposed region of the flow-cell contained four gold patches and fourinterstitial regions; the non-UV exposed region of the flow-cellcontained the remaining eight gold patches and eight interstitialregions.

FIG. 4B shows an image of the flow-cell following UV exposure. A plot ofgray value vs. distance is shown for lane 8 of the flow-cell in FIG. 4C.As shown in the plot, the signal from the four UV-exposed interstitialregions was reduced by about 50% compared to signal from the eight nonUV-exposed interstitial regions. This is indicative of photo-cleavage ofthe primers by UV light. The plot also shows that the signal fromprimers over the four UV-exposed gold patches was similar to the signalfor the eight non-UV exposed gold patches. This is indicative of thegold patches providing a mask protecting the primers fromphoto-cleavage.

These results show that metal patches located on the surface of asubstrate can form a near-field mask to radiation. Surprisingly, thenear-field mask provides effective protection even from non-collimatedlight. As demonstrated by these results, a near-field mask can be usedin combination with photo-cleavage to produce a surface having a patternof features containing nucleic acid primers.

Example III

This example describes methods that can be used to create an array ofnucleic acid features.

FIG. 5 provides a diagrammatic representation of two exemplary methodsfor creating an array of features, wherein the features contain a geland/or nucleic acids. Generally, a flow-cell is created as described inExamples I or II or elsewhere herein. The flow-cell surface has metalregions (such as gold or aluminum oxide) separated by interstitialregions (such as glass). A gel layer, such as polymerized silane-freeacrylamide (SFA), is present as a continuous coating over the flow-cellsurface. The flow-cell can also optionally include primers attached tothe gel layer. The flow-cell is irradiated from below, for example withUV light, such that the metal regions provide a near-field mask to blockirradiation of regions of the gel that are above the metal regions,whereas regions of the gel that are located above glass interstitialregions are irradiated. If primers are attached to the gel then themetal patches provide a near-field mask to block irradiation of primersthat are attached above the metal primers, whereas primers that arelocated above glass interstitial regions are irradiated.

In a first alternative the UV light causes polymerization of a gel. Forexample, as shown in FIG. 5 the UV light photo-initiates polymerizationof SFA at interstitial regions and the metal regions maskpolymerization. In this way an array of gel features is produced.

In a second alternative, UV light removes primers from interstitialregions. As shown in FIG. 5, the UV light photo-cleaves primers in theinterstitial regions while primers that are attached above the metalregions are masked from photo-cleavage. As such an array of nucleic acidfeatures is produced. Optionally, the primers are seeded with templatenucleic acids and the template nucleic acids are amplified, for example,by bridge amplification to form an array of nucleic acid clusters.

Further details regarding the first alternative are shown in FIG. 6. Theflow-cell surface is patterned to have circular glass regions surroundedby aluminum oxide. The aluminum oxide can be, for example, 40 nm thick.The glass regions have a diameter that is between 200 nm and 20 microns,but need not be limited to this range. The flow-cell has a fluidic spacethat is about 100 microns in height. A solution of silane-freeacrylamide and BRAPA having a photo-initiator moiety such as2,2′-Azobis[2-methyl-N-(2-hydroxyethyl)propionamide] or benzophenone isintroduced to the flow-cell. The flow-cell is irradiated with UV lightfrom below such that the metal regions provide a mask that preventspolymerization above the metal regions. However, UV light passes throughthe glass such that photo-induced polymerization occurs at each glassregion. Thus an array of circular features is formed, each featurecontaining polymerized SFA. Primers are grafted to the polymerized SFAand clusters are formed at the gel features using methods set forth inExamples I or II or elsewhere herein.

Further details regarding the second alternative are shown in FIG. 7.The flow-cell surface is patterned to have circular patches of aluminumoxide metal surrounded by glass interstitial regions. The aluminum oxidecan be, for example, 40 nm thick. The metal regions have a diameter thatis between 200 nm and 20 microns, but need not be limited to this range.The flow-cell has a fluidic space that is about 100 microns in height. Acoating of SFA polymer is formed to create a continuous layer over thesurface including the metal regions and the interstitial regions asdescribed in Example I. A primer is attached to the SFA layer via aphoto-cleavable linker as described in Example II. The flow-cell isirradiated with UV light from below such that the metal regions providea mask that prevents cleavage of primers above the metal regions.However, UV light passes through the glass such that photo-cleavage ofprimers occurs above each glass region. Thus an array of nucleic acidfeatures is formed, each feature containing a population of attachedprimers. Clusters are formed at the primer features using methods setforth in Examples I or II or elsewhere herein.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of these publicationsin their entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

What is claimed is:
 1. A method of making a nucleic acid array,comprising: (a) providing a surface having mask regions and transparentregions, the transparent regions having a composition that differs fromthe mask regions; (b) contacting the surface with a polymerizablematerial; (c) polymerizing the polymerizable material to form acontinuous gel layer on the surface, wherein the portions of the gellayer that coat the mask regions have greater mass than portions of thegel layer that coat the transparent regions; (d) contacting thecontinuous gel layer with a fluid comprising nucleic acids; (e)selectively and covalently attaching the nucleic acids to the gel layer,wherein the nucleic acids from the fluid that are attached to portionsof the gel layer that coat the mask regions are at a greater densitythan the nucleic acids that are attached to portions of the gel layerthat coat the transparent regions; and (f) selectively photochemicallycleaving the nucleic acids from the gel layer in the transparentregions.
 2. The method of claim 1, wherein the nucleic acids compriseprimer sequences.
 3. The method of claim 2, further comprisinghybridizing template nucleic acids to the nucleic acids attached to thegel layer.
 4. The method of claim 1, wherein the mask regions comprisemetal regions.
 5. The method of claim 1, wherein the transparent regionson the surface form interstitial regions that completely surround themask regions on the surface.
 6. The method of claim 1, wherein thecontacting occurs under conditions wherein a uniform concentration ofthe nucleic acids contacts the portions of the continuous gel layer thatcoat the mask regions and the transparent regions.
 7. The method ofclaim 6, wherein the attaching occurs while the uniform concentration ofthe nucleic acids contacts the portions of the continuous gel layer thatcoat the mask regions and the transparent regions.
 8. The method ofclaim 1, wherein the surface is located in a flowcell.
 9. The method ofclaim 1, wherein the continuous gel layer comprises agarose, gelatin, ora polyacrylamide polymer.
 10. The method of claim 1, wherein thetransparent regions comprise glass, plastic, or silica.
 11. The methodof claim 1, wherein the nucleic acids comprise a photocleavable linker.12. The method of claim 1, wherein the gel material is polymerizedsilane-free acrylamide (SFA) and is formed by polymerization ofsilane-free acrylamide and N—(S-bromoacetamidylpentyl)acrylamide(BRAPA).
 13. The method of claim 1, wherein the polymerizing isaccomplished by way of a temperature-dependent change in state.
 14. Themethod of claim 1, wherein the polymerizable material is acrylamide,methacrylamide, hydroxyethyl methacrylate, or N-vinyl pyrrolidinone, orderivatives or co-polymers thereof.
 15. The method of claim 1, whereinselectively photochemically cleaving the nucleic acids from the gellayer in the transparent regions involves: positioning the surface overa light source; and turning on the light source, whereby the maskregions mask light from for the nucleic acids attached to the gel layerthat coat the mask regions and the nucleic acids attached to the gellayer that coat the interstitial regions are exposed to the light.
 16. Amethod of making a nucleic acid array, comprising: (a) providing asurface having mask regions and transparent regions, the transparentregions having a composition that differs from the mask regions; (b)contacting the surface with a polymerizable material; (c) polymerizingthe polymerizable material to form a continuous gel layer on thesurface, wherein the portions of the gel layer that coat the maskregions have greater mass than portions of the gel layer that coat thetransparent regions; (d) contacting the continuous gel layer with afluid comprising nucleic acids; (e) selectively and covalently attachingthe nucleic acids to the gel layer, wherein the nucleic acids from thefluid that are attached to portions of the gel layer that coat the maskregions are at a greater density than the nucleic acids that areattached to portions of the gel layer that coat the transparent regions;and (f) selectively inactivating the nucleic acids that are attached tothe portions of the gel layer that coat the transparent regions comparedto the nucleic acids that are attached to the portions of the gel layerthat coat the mask regions.
 17. The method of claim 3, furthercomprising amplifying the template nucleic acids on the gel layer. 18.The method of claim 17, wherein the amplifying comprises bridgeamplifying to form nucleic acid clusters attached to the gel layer. 19.The method of claim 16, wherein selectively inactivating the nucleicacids that are attached to the portions of the gel layer that coat thetransparent regions involves exposing the nucleic acids that areattached to the portions of the gel layer that coat the transparentregions to photolysis or laser ablation, and wherein the mask regionsmask the nucleic acids attached to the gel layer that coat the maskregions from the photolysis or laser ablation.
 20. The method of claim16, wherein the mask regions comprise metal regions, and wherein thetransparent regions comprise glass, plastic, or silica.
 21. The methodof claim 16, wherein the continuous gel layer comprises agarose,gelatin, or a polyacrylamide polymer.
 22. The method of claim 16,wherein the polymerizable material is acrylamide, methacrylamide,hydroxyethyl methacrylate, or N-vinyl pyrrolidinone, or derivatives orco-polymers thereof.
 23. The method of claim 16, wherein the nucleicacids comprise primer sequences.
 24. The method of claim 16, wherein thetransparent regions on the surface form interstitial regions thatcompletely surround the mask regions on the surface.